Field of the Invention
Compositions and methods are provided for modifying the expression of genes. More particularly, synthetic RNA structures for silencing genes through RNA interference (RNAi), and methods of using those synthetic structures for silencing genes of interest are provided.
Description of Related Art
RNA interference (RNAi) is the process of post-transcriptional repression of a target gene by small non-coding RNAs. The biogenesis of these small microRNAs from long primary transcripts yields RNAs which are loaded on to the RNA-induced silencing complex (RISC), which can bind to target RNA and catalyze its endonucleolytic cleavage thereby turning over the target transcript for degradation. The process of inducing RNAi with synthetic short-interfering RNAs (siRNAs) is traditionally initiated by delivery of precursor RNAs which are further processed by the enzyme Dicer, an endonuclease in the RNAi pathway, and then the resulting RNA strand, typically 20-25 residues long, is loaded into RISC.
The two main drawbacks to this standard approach are the significant RNA degradation observed over time in serum and the significant challenges of delivery. To address the stability issues, modified duplexes of the siRNAs which exhibit nuclease resistance and potent RNAi activity are introduced into cells. However, therapeutic delivery of these larger RNA constructs is challenging.
Mature miRNAs are short RNAs which can be easily synthesized in the solid-phase and thus researchers have used small synthetic RNAs to induce an RNAi response. These small-interfering RNAs (siRNAs) are generally delivered as duplexes and exhibit powerful gene interference. The main drawback of the use of siRNAs in RNAi is that without a steady source of the siRNAs, the transfected RNAs quickly succumb to cellular RNases and thus their effect is generally short-lived. This has driven researchers to develop modifications to these siRNAs that both lengthen the interference effect and improve their potency. The types of modifications range from backbone modifications that provide RNase stability, sugar modifications to improve siRNA binding and RNAse stability and base modifications to provide increased affinity and target selectivity. Additionally, the use of siRNA mimics, siRNA conjugates or delivery vehicles has been reported to induce powerful RNAi responses. However the use of some modifications, including phosphorothioates, 2′-OMe or 2′-fluoro, can lead to undesired side-reactions or affected pharmacokinetic properties.
Recent reports suggest that most miRNAs derive from non-coding regions of genes, with nearly eighty percent of mouse and human miRNAs originating in introns of mRNA-coding genes. These mirtron sequences are located as isolated sequences flanked by exons, and thus require efficient splicing for their excision from the main transcript sequence. The highly conserved splice site residues, once spliced and debranched by debranching enzyme (Dbr), generate a hairpin similar to that generated by Drosha. Therefore, these pre-miRNA transcripts require Dbr activity but do not require Drosha activity for maturation. Following Dbr debranching, mirtron-derived pre-miRNAs are exported and processed like canonical miRNAs. However, these alterations to the traditional miRNA pathway do not address several issues, such as duration of the effect.